Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.
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ABSTRACT:
SUBMITTER: Stadtfeld M
PROVIDER: S-EPMC3538379 | biostudies-literature | 2008 Mar
REPOSITORIES: biostudies-literature
Publications
Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.
Stadtfeld Matthias M Maherali Nimet N Breault David T DT Hochedlinger Konrad K
Cell stem cell 20080214 3
Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition ...[more]
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Project description:Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals.
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Project description:This SuperSeries is composed of the SubSeries listed below.
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Project description:Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation.
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